Use of outer membrane protein A (OMPA) in treatment/prevention/diagnosis of infections caused by Klebsiella pneumoniae and other gram-negative bacteria

ABSTRACT

The present invention provides a method for the treatment and/or prevention of bacterial infection caused by  Klebsiella pneumoniae  and other gram-negative bacteria in central nervous system and/or peripheral blood circulation in a mammal by administering effective amount of outer membrane protein A (OmpA) or its derivatives to a mammal. Also provided are a method for vaccinating a mammal to produce an antibody against bacterial infection caused by  Enterobacteriaceae  family in central nervous system and/or peripheral blood circulation and a method of detecting or diagnosing bacterial infections caused by  Enterobacteriaceae  family in central nervous system and/or peripheral blood circulation in a mammal.

FIELD OF THE INVENTION

The present invention is directed to a method for the treatment and/orprevention and/or diagnosis of bacterial infection caused by Klebsiellapneumoniae and other gram-negative bacteria in central nervous systemand/or peripheral blood circulation in a mammal by administering aneffective amount of outer membrane protein A or its derivatives to amammal.

BACKGROUND OF THE INVENTION

In general, the central nervous system (CNS) is well defended againstinfection. The spine and brain are sheathed in tough, protectivemembranes. The outermost membrane, the dura mater, and the next layer,the arachnoid, entirely encase the brain and spinal cord. However, thesedefenses are not absolute. In some cases, bacteria gain access to areaswithin the CNS. Bacterial infections can be pyogenic infections (e.g.,meningitis; brain abscess; subdural and epidural abscesses),tuberculosis, neurosyphilis, or leprosy. Typically, bacterial invasionresults from the spread of a nearby infection; for example, a chronicsinus or middle ear infection can extend beyond its initial site.Bacteria may also be conveyed to the CNS from distant sites of infectionby the bloodstream. In rare cases, head trauma or surgical proceduresmay introduce bacteria directly into the CNS. However, the source ofinfection cannot always be identified.

The goal of treatment of a bacterial infection is to stop the infection,relieve symptoms, prevent complications, and, if necessary, provide lifesupport. A two-pronged approach is taken to treat bacterial infections.First, antibiotic therapy against an array of potential infectiousbacteria is begun. The second stage involves surgery to drain theinfected site. Once the bacterial species is identified, drug therapycan be altered to a more specific antibiotic. However, surgery may notbe an option in some cases, such as when there are numerous sites ofinfection or when infection is located in an inaccessible area of thebrain.

Outer membrane protein A (OmpA) was initially described by Henning andcoworkers in 1975. It has 325 amino acid residues and exhibitsheat-modifiable electrophoretic mobility on SDS-PAGE. The N-terminaldomain of OmpA is comprised of 177 amino acids and is believed totraverse the outer membrane eight times. OmpA is involved in maintainingthe shape of bacteria, serves as a phage receptor and a receptor forF-mediated conjugation, and has limited pore-forming properties. OmpAenhances uptake of LPS into macrophages and has been reported to beinvolved in E. coli invasion of the central nervous system. WO 9201001provides a method for producing pure cloned outer membrane proteins, andto provide a method for their renaturation so as to regain biologicallyor immunologically active epitopes which are capable of eliciting theproduction of antibodies in animals. Pascale Jeannin et al. reports thatouter membrane protein A (OmpA) is a class of protein highly conservedamong the Enterobacteriaceae family throughout evolution and OmpAappears as a new type of pathogen-associated molecular pattern (PAMP)usable as a vector in anti-infectious and therapeutic anti-tumorvaccines to elicit CTLs (Vaccine, Volume 20, Supplement 4, 19 Dec. 2002,pages A23-A27).

However, there are no reports relating to the new use of an outermembrane protein A and its derivatives in the treatment and/orprevention and/or diagnosis of bacterial infection in central nervoussystem and/or peripheral blood circulation.

SUMMARY OF THE INVENTION

An object of the invention is to provide a method for the treatmentand/or prevention of bacterial infection caused by Enterobacteriaceaefamily in central nervous system and/or peripheral blood circulation ina mammal, which comprises administering to said mammal an effectiveamount of an outer membrane protein A (OmpA) from Enterobacteriaceaefamily

Another object of the invention is to provide a method for vaccinating amammal to produce an antibody against bacterial infection caused byEnterobacteriaceae family in central nervous system and/or peripheralblood circulation, which comprises administering to said mammal aneffective amount of an OmpA from Enterobacteriaceae family.

A further object of the invention is to provide a method of detecting ordiagnosing bacterial infections caused by Enterobacteriaceae family incentral nervous system and/or peripheral blood circulation in a mammal,which comprises coating a first specific anti-OmpA antibody onto amatrix surface that can immunospecifically bind to OmpA molecule inblood or OmpA on bacterial membrane, adding a sample from peripheralblood circulation and/or the central nervous system to the matrix,adding a second anti-OmpA antibody with a label, and detecting thebinding of the anti-OmpA antibodies to the OmpA molecule or OmpA onbacterial membrane, wherein the binding result indicates that the mammalmay suffer from the bacterial infections in the peripheral bloodcirculation and/or the central nervous system, and wherein the OmpA isfrom Enterobacteriaceae family.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course of E. coli strain adhesion.

FIG. 2 shows the time course of E. coli strain invasion.

FIG. 3 shows the inhibition of E. coli E 44 invasion into C6 gliomacells by OmpA.

FIG. 4 shows the survival of C57BL/6 mice intracerebrally injected withE. coli strains.

FIG. 5 shows that OmpA prolongs the survival of the mice intracerebrallyinjected with E. coli E44.

FIG. 6 shows that OmpA prolongs the survival of the mice intracerebrallyinjected with Klebsiella pneumoniae.

FIG. 7 shows that OmpA prolongs the survival of the mice intracerebrallyinjected with Salmonella typhi.

FIG. 8 shows the binding of anti-OmpA IgY antibodies to E. coli E44using Confocal Spectral Microscope Imaging System.

FIG. 9 shows the electrophoresis plot of the antibody against OmpA.

DETAILED DESCRIPTION OF THE INVENTION

The invention unexpectedly found that the outer membrane protein A(OmpA) can be used to treat and/or prevent and/or diagnose bacterialinfection in central nervous system and/or peripheral blood circulation.In addition, an antibody binding to OmpA has been developed to assayOmpA levels in a biological sample and to detect or diagnose bacterialinfection in central nervous system and/or peripheral blood circulation.

The invention provides a method for the treatment and/or prevention ofbacterial infection in central nervous system and/or peripheral bloodcirculation in a mammal, which comprises administering to said mammal aneffective amount of an outer membrane protein A or its derivatives.

The “central nervous system” used herein denotes to that part of thenervous system that consists of the brain and spinal cord. The“peripheral blood circulation” denotes to the blood in the systemiccirculation.

The “bacterial infection” used herein denotes the infection caused byGram-negative bacteria. Preferably, the bacterial infection is caused byEnterobacteriaceae or other Gram-negative bacteria. More preferably, thebacterial infection is caused by Shigella, Salmonella, Klebsiella,Escherichia, Citrobacter, Enterobacter or Serratia. Most preferably, thebacterial infection is caused by Escherichia coli, Klebsiellapneumoniae, Salmonella typhi, Enterobacter aerogenes.

The “outer membrane protein A (OmpA)” used herein denotes to any OmpAfrom Gram-negative bacteria and any recombinant OmpA. OmpA is anabundant structural protein of the outer membrane of Gram-negativebacteria. The “OmpA derivatives” denotes to proteins derived from OmpA,which have same function as that of OmpA. For example, OmpA recombinantshaving same function with OmpA are OmpA derivatives. Preferably, OmpA orits derivative is obtained from Enterobacteriaceae or otherGram-negative bacteria. OmpA is a class of protein highly conservedamong the Enterobacteriaceae family (see Vaccine, Volume 20, Supplement4, 19 Dec. 2002, pages A23-A27), so persons skilled in the art recognizethat OmpA from various bacteria may have the same function. Nguyen T Net al. finds that after alignment, the amino acid sequences of OmpAs ofEscherichia coli, Klebsiella pneumoniae, Salmonella typhi, Enterobacteraerogenes have high similarity (Gene 1998 210:93). More preferably, OmpAor its derivative is obtained from Shigella, Salmonella, Klebsiella,Escherichia, Citrobacter, Enterobacter or Serratia. Most preferably,OmpA or its derivative is obtained from Escherichia coli, Klebsiellapneumoniae or Salmonella typhi, Enterobacter aerogenes. According to oneembodiment of the invention, a recombinant E. coli OmpA can effectivelytreat an infection caused by Escherichia coli, Klebsiella pneumoniae,Salmonella typhi, or Enterobacter aerogenes.

According to the invention, a therapeutically effective amount of OmpAor its derivatives of the invention can be administered to a mammal,including a human or non-human mammal, suffering from bacterialinfection in both central nervous system and/or peripheral blood system.According to the invention, the administration of OmpA or itsderivatives of the invention can be carried out in various ordinaryways. Administration forms suitable for oral administration are thosewhich function according to the state of the art and deliver OmpA or itsderivatives of the invention in a rapid and/or modified way, forexample, tablets (uncoated or coated tablets, for example with coatingswhich are resistant to gastric juice or dissolve slowly or are insolubleand which control the release of the compound of the invention), tabletswhich rapidly disintegrate in the mouth, or films/wafers,films/lyophilisates, capsules (for example hard or soft gelatincapsules), sugar-coated tablets, granules, pellets, powders, emulsions,suspensions, aerosols or solutions.

Parenteral administration includes intravenous drip or infusion,subcutaneous, intraperitoneal or intramuscular injection, pulmonaryadministration, e.g., by inhalation or insufflation, or intrathecal orintraventricular administration. Administration forms suitable forparenteral administration are, inter alia, injection and infusionpreparations in the form of solutions, suspensions, emulsions,lyophilisates or sterile powders.

Oral or parenteral administration is preferred, especially oral andintravenous administration. Intravenous dosage is particularly preferredfor example for the treatment of acute central nervous system infection.

OmpA or its derivatives used according to the invention can be convertedinto suitable pharmaceutical compositions. This can take place in aknown manner by mixing with inert, non-toxic, pharmaceutically suitableexcipients. These excipients include, inter alia, carriers (for examplemicrocrystalline cellulose, lactose, mannitol), solvents (e.g. liquidpolyethylene glycols), emulsifiers and dispersants or wetting agents(for example sodium dodecyl sulfate, polyoxysorbitan oleate), binders(for example polyvinylpyrrolidone), synthetic and natural polymers (forexample albumin), stabilizers (e.g. antioxidants such as, for example,ascorbic acid), colors (e.g. inorganic pigments such as, for example,iron oxides) and masking tastes and/or odors.

The administration form of the invention comprises 0.0001% to 10% byweight of OmpA or its derivatives, preferably 0.5% to 5% by weight ofOmpA or its derivatives. The time of treatment with the pharmaceuticalcomposition of the invention is determined on the basis of severity ofthe disease to be treated and the conditions of individual patients. Adoctor shall determine the adequate amount of time of treatment with thepharmaceutical composition of the invention.

The invention also provides a method for vaccinating a mammal to producean antibody against bacterial infection in central nervous system and/orperipheral blood circulation, which comprises administering to saidmammal an effective amount of an outer membrane protein A or itsderivatives. In view of the discovery that OmpA can treat and/or preventand/or diagnose bacterial infection in central nervous system and/orperipheral blood circulation, OmpA was introduced to an animal forvaccination. It is found that OmpA can induce specific antibodyproduction.

The antibodies of the invention can be produced by any method known inthe art. Polyclonal antibodies to OmpA can be produced by variousprocedures well known in the art. For example, OmpA can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for OmpA. Various adjuvants may be used to increasethe immunological response, depending on the host species, and includebut are not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

Antibodies of the invention can be used to assay OmpA levels in abiological sample using classical serological and immunohistologicalmethods as described herein or as known to those of skill in the art.Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

The invention further provides a method of detecting or diagnosingbacterial infection in central nervous system and/or peripheral bloodcirculation in a mammal. In one embodiment, the detection or diagnosiscomprises: coating a first specific anti-OmpA antibody onto a matrixsurface (such as ELISA plates or magnetic nano-particles) that canimmunospecifically bind to OmpA molecules in blood or OmpA on bacterialmembranes, adding a sample from peripheral blood circulation and/or thecentral nervous system to the matrix, adding a second anti-OmpA antibodywith a label, and detecting the binding of the anti-OmpA antibodies tothe OmpA molecules or OmpA on bacterial membranes, wherein the bindingresult indicates that the mammal may suffer from the bacterialinfections in the peripheral blood circulation and/or the centralnervous system.

According to the invention, the first specific anti-OmpA antibody iscoated onto the matrix surface using a method and commercial coatingbuffer known in the art and any appropriate matrix can be used in themethod. The matrix is preferably ELISA plates or magneticnano-particles. The anti-OmpA antibody can specifically bind to OmpAmolecules in blood or OmpA on bacterial membranes. To allow the bindingdetectable, the anti-OmpA antibody specifically binding to OmpAmolecules can be detected by using a second anti-OmpA antibody with alabel. According to the invention, the term “label” refers to a moleculeor moiety having a property or characteristic which is capable ofdetection. A label may be directly detectable, as with radioisotopes,fluorophores or chemilumiphores; or a label may be indirectlydetectable, as with haptens or polynucleotide tails. When indirectlabels are used for detection or signaling purposes, they are used inconjunction with a signaling entity complex. A “signaling entity” is amolecule or moiety which provides the detectable property orcharacteristic. The signaling entity may be direct, as with a colloidalparticle (e.g. colloidal gold or selenium); or it may be indirect, aswith an enzyme (e.g. alkaline phosphatase, beta.-galactosidase orhorseradish peroxidase). Indirect signaling entities may requireadditional components, e.g. substrate, as is well known in the arm. The“signaling entity complex” includes a signaling entity conjugated tospecific binding partner, such as an antibody or polynucleotide. Suchconjugates may be prepared according to any known method of conjugation.

According to the invention, the binding of the anti-OmpA antibody to theOmpA molecule in peripheral blood or central nervous system can be usedto detect the existence of the OmpA molecule. If the OmpA moleculeexists in the peripheral blood or central nervous system of a subject,it represents that the subject may be infected by a bacteria with OmpAmolecule.

EXAMPLE

The following examples illustrate the invention. The invention is notrestricted to the examples.

Example 1 Adhesion and Invasion Assay

For total C6 glioma cell-associated bacteria studies, confluent cellmonolayers were incubated with respective bacteria strain E. coli E44,E91 and MG1655 at indicated time intervals at 37° C. E. coli E44 is a K1strain RE218 (O18:K1:H7) and derived from a cerebrospinal fluid of aneonate with meningitis. E. coli E91 is a mutant lacking the entire OmpAgene and generated from strain E44. E. coli MG1655 is a nonpathogenicstrain and non-invasive in blood brain barrier, so it was used as acontrol.

In the adhesion assay, the C6 glioma cells were infected with theabove-mentioned bacteria strains (MOI (bacteria-to-cell ratio)=10) atindicated time intervals. The C6 glioma cell monolayers were then washedwith culture medium three times and lysed in 0.5% Triton X-100. Thereleased bacteria were enumerated by plating on sheep blood agar plates.As shown in the results of the adhesion assay (FIG. 1), the numbers ofE. coli E44 largely increases over time. However, the numbers of E. coliE91 and MG1655 are smaller than E. coli E44.

In the invasion assay (gentamicin protection assay), for intracellularbacteria studies, the glioma C6 confluent cell monolayers were incubatedwith the above-mentioned bacteria strain (MOI=10) at indicated timeintervals at 37° C. The monolayers were then washed with culture mediumthree times and further incubated with culture medium containinggentamicin (100 μg ml⁻¹) for 2 hours to kill extracellular bacteria. Themonolayers were washed three times again and lysed in 0.5% Triton X-100.The released bacteria were enumerated by plating on sheep blood agarplates. As shown in FIG. 2 for the invasion assay, the numbers of E.coli E44 are much larger than those of E. coli E91 and MG1655. In theinvasion inhibition assay, the glioma C6 confluent cell monolayers wereinfected with E. coli E44 (MOI=10) with 4 μg or 40 μg OmpA for 2 hours.The monolayers were then washed with culture medium three times andfurther incubated with culture medium containing gentamicin (100 μgml⁻¹) for 2 hours to kill extracellular bacteria. The released bacteriawere enumerated by plating on sheep blood agar plates. As shown in FIG.3, the mixtures of E. coli E44 and OmpA indeed inhibit the invasion ofE. coli E44. OmpA can inhibit more than 55% (4 μg) and 80% (40 μg)invasion of E. coli E44.

Example 2 Animal Experiments

C57BL/6 mice were obtained from the National Laboratory Animal Center ofTaiwan, and kept under pathogen-free conditions. Animal procedures wereperformed in accordance with the institutional protocol of TaipeiMedical University under an approved protocol.

8-12 week-old C57BL/6 mice were randomly divided in groups. Each groupcontained 5 to 10 mice. Mice were anesthetized with pentobarbital sodiumsalt (50 mg kg-1) by intraperitoneal injection, and then each mouse wasinfected with 5×10⁵ E. coli strains (E44, E91, or MG1655 in 20 μl PBS,or 5 μg LPS in 20 μl PBS by intracerebral injection. 20 μl PBS was usedas a control treatment. Survival in C57BL/6 mice after E. coli strainsinfection was assessed 8 days post administration. As shown in FIG. 4,all of the mice infected with E. coli E44 died after 2 days.

To investigate the role of recombinant OmpA in the survival of C57BL/6mice following intracerebral E44 administration, 8-12 weeks-old C57BL/6mice were anesthetized with pentobarbital sodium salt (50 mg kg⁻¹) byintraperitoneal injection. Then each mouse was infected with E44 5×10⁵in 20 μl PBS, without or with premixed with 8 μg or 20 μg recombinant E.coli OmpA, by intracerebral injection. PBS 30 μl was used as a controltreatment. Survival in C57BL/6 mice was assessed 8 days postadministration. FIG. 5 showed that OmpA prolongs the survival of themice intracerebrally injected with E. coli E44. Around 40% and 80% ofthe mice survived in E. coli E44 with 8 μg OmpA and E. coli E44 with 20μg OmpA.

The above-mentioned mice experiments were performed for the infectioncaused by Klebsiella pneumoniae or Salmonella typhi and the experimentalconditions and steps used were the same as those for E. coli except thatonly 20 μg recombinant E. coli OmpA was used. FIGS. 6 and 7 show thatOmpA prolonged the survival of C57BL/6 mice infected with K. pneumoniaeand S. typhi. Around 80% of the mice survived when K. pneumoniae wastreated with 20 μg E. coli OmpA. Similarly, when S. typhi was treatedwith 20 μg E. coli OmpA, the survival of mice was significantlyprolonged.

Example 3 Immunocytochemistry

C6 glioma cell monolayers were infected with E. coli and fixed with 4%paraformaldehyde. The resulting C6 cell samples were blocked with 1% BSAand then incubated with a solution containing polyclonal chickenanti-OmpA IgY Ab (diluted 1:100 and 1:1000) from chicken so that theanti-OmpA IgY Ab could bind to the OmpA of E. coli. Polyclonal IgY Abfrom non-immunized chicken was used as a negative control. Finally,samples were incubated with FITC-labeled anti-IgY secondary antibody(diluted 1:500). Slides were mounted in 50% glycerol-PBS, and thenexamined with TCS SP5 Confocal Spectral Microscope Imaging System(Leica). FIG. 8 showed that the anti-OmpA antibodies produced clearlybind to E44, whereas IgY from non-immunized chicken did not show anybinding activity.

Example 4 Immunization Assay

C57BL/6 mice used in this assay are the same as those in Example 2. Atthe first week, the mice were immunized with 30 μg OmpA with completeFreund's adjuvant as the first immunization. At the second week, 30 μgOmpA with incomplete Freund's adjuvant as the second immunization. Atthe third week, 30 μg OmpA with incomplete Freund's adjuvant as thethird immunization. At the fourth week, 30 μg OmpA with incompleteFreund's adjuvant as the fourth immunization. Thereafter, the polyclonalantibodies from the immunized mice were isolated and used to detect theOmpA molecule immobilized on the nitrocellulose paper. FIG. 9 showedthat OmpA molecule can be detected using the polyclonal antibodieselicited in the mice in a western blot analysis.

1. A method for the treatment of bacterial infection caused by K.pneumoniae or S. typhi in central nervous system and/or peripheral bloodcirculation in a mammal, which comprises administering to said mammal aneffective amount of an outer membrane protein A (OmpA) from E. coli, K.pneumoniae or S. typhi.
 2. The method of claim 1, wherein the mammal ishuman.
 3. The method of claim 1, wherein the OmpA is a recombinant OmpA.4. The method according to claim 1, wherein the OmpA is administratedorally or via intravenous injection.
 5. The method according to claim 1,wherein the OmpA is administrated at an amount of 0.0001% to 10% byweight of OmpA.
 6. The method according to claim 1, wherein the OmpA isadministrated at an amount of 0.5% to 5% by weight of OmpA.
 7. A methodfor vaccinating a mammal to produce an antibody against bacterialinfection caused by K. pneumoniae or S. typhi in central nervous systemand/or peripheral blood circulation, which comprises administering tosaid mammal an effective amount of an OmpA from E. coli, K. pneumoniaeor S. typhi.
 8. The method of claim 7, wherein the antibody is apolyclonal antibody.
 9. The method of claim 7, wherein the mammal ishuman.
 10. The method of claim 7, wherein the OmpA is a recombinantOmpA.
 11. The method according to claim 7, wherein the OmpA isadministrated at an amount of 0.0001% to 10% by weight of OmpA.
 12. Themethod according to claim 7, wherein the OmpA is administrated at anamount of 0.5% to 5% by weight of OmpA.
 13. A method of detecting ordiagnosing bacterial infections caused by K. pneumoniae or S. typhi incentral nervous system and/or peripheral blood circulation in a mammal,which comprises coating a first specific anti-OmpA antibody onto amatrix surface that can immunospecifically bind to OmpA molecule inblood or OmpA on bacterial membrane, adding a sample from peripheralblood circulation and/or the central nervous system to the matrix,adding a second anti-OmpA antibody with a label, and detecting thebinding of the anti-OmpA antibodies to the OmpA molecule or OmpA onbacterial membrane, wherein the binding result indicates that the mammalmay suffer from the bacterial infections in the peripheral bloodcirculation and/or the central nervous system, and wherein the OmpA isfrom E. coli, K. pneumoniae or S. typhi.
 14. The method of claim 13,wherein the mammal is human.
 15. The method of claim 13, wherein thematrix is ELISA plates or magnetic nano-particles.
 16. The method ofclaim 13, wherein the label is radioisotopes, fluorophores orchemilumiphores.